DESCRIPTION: Injectable stem cell therapy is a promising, minimally invasive strategy to treat a wide range of injuries and degenerative diseases. Current stem cell-based clinical trials to treat cardiovascular diseases such as peripheral arterial disease (PAD) have generally shown limited efficacy, in part due to poor cell survival. We have previously demonstrated using animal models of PAD that the survival of human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) declines rapidly after injection into ischemic tissue, leading to only a modest improvement in blood perfusion recovery. To address this limitation of cell survival, we have previously engineered hydrogels that can be co-injected to protect cells from mechanical membrane damage during syringe-needle injection. However, such hydrogels are very compliant (G' ~10 Pa), and not suitable for many biomedical applications. Therefore, we propose to develop Mixing-Induced Two-Component Hydrogels modified with polyethylene glycol and poly(N-isopropylacrylamide) (MITCH-PEG-PNIPAM). Our goal is to engineer hydrogels that provide tunable mechanical stiffness and sustained delivery of pro-survival factors to inhibit hypoxia-induced apoptosis while still providing significant membrane protection during injection. Accordingly, in Specific Aim 1, we will evaluate the hypothesis that tuning of th hydrogel rigidity and the release kinetics of pro-survival factors will significantly improve the viability of stem cells exposed to injection flow and hypoxia. Human iPSC-ECs will be encapsulated within the engineered hydrogels of varying stiffness (G'= 10-100 Pa) and pro-survival factors (Rho-associated kinase inhibitor Y-27632; and insulin-like growth factor-1). Cells will be subjected to an in vitro model of injection and acutely assayed for membrane damage, mitochondrial activity, metabolic activity, and apoptotic markers. At 7, 14, and 28 days post-injection, cell proliferation rate, metabolic activity, apoptotic markers, and EC phenotype will be quantified in both normoxic (20% O2) and hypoxic (1% O2) culture conditions to mimic in vivo ischemia. Based on the results of these assays, we will choose the hydrogel stiffness and pro-survival factor that maximizes cell survival. In Specific Aim 2 we will validate the in vitro resuls in NOD-SCID mice with induced hindlimb ischemia, an experimental model of PAD, by evaluating the efficacy of the hydrogel in enhancing cell survival and therapeutic efficacy of the cells. Cells will be encapsulated in the optimal hydrogel and pro-survival factors before injection into the ischemic limb. Controls include saline injection (with and without cells, with and without pro-survival factors) and hydrogel injection (without cells, with and without pro-survival factors) Cell survival and blood perfusion will be tracked noninvasively by bioluminescence imaging and laser Doppler spectroscopy, respectively. Histological explants will be analyzed for necrosis, inflammation, neovascularization, tissue regeneration, and presence of transplanted cells. The results of the proposed studies will lead to a new biomaterials approach to enhance the efficacy of stem cell therapy for clinical applications.